The relation between the genotype and the phenotype of an organism is fundamental to biology. Based on the annotated genome sequence and biochemical information it is possible to reconstruct genome-scale metabolic maps, and in silico methods have been developed to interpret and predict the optimal utilization of the metabolic map under a given growth condition. Using the defined Escherichia coli MG1655 metabolic genotype, experimentally testable hypotheses have been formulated in silico describing the quantitative relation between the uptake rate of a primary carbon source, oxygen uptake rate, and cellular growth. Experiments with E. coli have been performed and experimental data were found to be consistent with the optimal use of the metabolic network to support growth of E. coli under the experimental conditions considered, which were growth on acetate, succinate, and malate. These important results form the basis for the proposed program. It is comprised of three parts, 1) to continue the generation of experimentally testable hypotheses for a broader ranger of carbon sources and to profile the gene expression patterns for these growth conditions, 2) to generate 40 knockout strains and perform both growth experiments and gene expression studies to characterize their metabolic behavior and 3) to perform evolution experiments for wild-type and knockout strains of E. coli that exhibit sub-optimal growth phenotypes. Additionally, gene expression studies will be performed for these evolving strains during and after evolution to identify the molecular events that are elicited during the evolutionary process. Hence the experimental program has the specific aims of determining optimal phase plane behavior for a range of growth conditions, and to determine if metabolic gene expression patterns are consistent with predicted pathway used for both wild-type and knockout strains. If the program is successfully implemented and executed we will have refined and improved our understanding of the metabolic genotype-phenotype relationship for E. coli under the conditions examined, an accomplishment of clear fundamental significance.